Clostridium difficile is the leading cause of nosocomial, antibiotic-associated diarrhea in the United States. C. difficile is a Gram-positive, spore-forming anaerobic bacterial pathogen. In a typical infection, susceptible individuals ingest C. difficile spores from contact with contaminated surfaces. These spores germinate into actively growing vegetative cells in the colon of the host. C. difficile vegetative cells secrete to protein toxins essential to virulence. These toxins, TcdA and TcdB, promote actin depolymerization, host cell death, and inflammation. Colonization is a requisite step to C. difficile infection and disease, yet the surface structures that mediate adherence to the intestina epithelium are incompletely understood. The flagellar apparatus contributes to motility and adherence to the intestinal epithelium in epidemic C. difficile strains. Recent evidence demonstrates that flagellar gene transcription positively impacts toxin production, further emphasizing the importance of flagella in diarrheal disease. However, phenotypic variability of flagellum production is readily observed in C. difficile strains under standard laboratory conditions. How C. difficile regulates expression of flagellar biosynthesis genes and the impact of this regulation on virulence are largely unknown. Our recent studies demonstrate that flagellum production in C. difficile is phase variable. A DNA sequence flanked by inverted repeats upstream of the early flagellar operon undergoes DNA inversion. One orientation of the DNA sequence results in C. difficile that is flagellated and toxigenic; the other orientation resuls in non-motile, non-toxigenic bacteria. Our working hypothesis is that site-specific recombination controls flagellar gene expression in C. difficile (Specific Aim 1), and phase variable flagellum production contributes to evasion of host innate immune detection to allow less restricted bacterial growth in the host intestine (Specific Aim 2). For Specific Aim 1, we will employ techniques in molecular biology, bacterial genetics, and protein biochemistry to determine how the orientation of the flagellar switch controls gene expression, identify the recombinase(s) mediating inversion, and determine the role of the inverted repeats. For Specific Aim 2, we will combine cell culture and animal infection models to characterize the spatial and temporal distribution of flagellar phase variants, evaluate the role of membrane-bound and cytosolic pathogen recognition receptors in detection of flagellar phase variants, and assess the role of flagellar phase variation in a primary infection model. We propose that C. difficile represses flagellum and toxin production upon initial adherence to the epithelium to enable bacterial growth without promoting host clearance mechanisms. These studies could facilitate the discovery of novel anti-virulence drug targets for emerging drug resistant C. difficile strains, directly supporting the mission of the NIAID to better understand, treat, ultimately prevent infectious, immunologic and allergic disease.